1. Field of the Invention
The present invention relates to a preparation method of a biotinylated protein and to a method for detecting a substance interacting with a biotinylated protein, using the protein. More particularly, the invention relates to a detection method wherein a biotinylated protein is prepared in a cell-free protein synthesizing system, and a substance interacting with the protein is detected.
This application claims the priority of Japanese Patent Application Nos. 2006-182785 and 2005-377840, which are incorporated herein by reference.
2. Related Background of the Invention
A variety of methods are utilized for analyzing a biomolecule interaction in an intracellular reaction. Such methods include, for example, ELISA (Enzyme Linked Immunosorbent Assay), DELFIA (Dissociation Enhanced Lanthanide Fluoroimmunoassay), and SPA (Scintillation Proximity Analysis). These methods are referred to as so-called “heterogeneous assay,” and used in post-genome researches such as Proteomics, Functional genomics. According to these methods, however, in a detection step, one or more washing operations are required, thus these methods have defects in sample-processing capacity, and the like.
In order to solve the above-mentioned defects, homogeneous (or mix-and-measure) assay technologies have been developed. In the assay technologies, the measurement is performed throughout in a solution state (homogeneous system), and no washing steps are required, but highly accurate data can be obtained. Additionally, since they do not require a solid phase, it is easy to be downsized, which greatly contributes to saving of precious regents, cost reduction, and economy of effort. Typical examples thereof include, for example FRET, BRET, EFC, SPA, FP, and ALPHA.
Of the above-mentioned homogeneous assays, ALPHASCREEN™ commercially available from Perkin-Elmer Corp., which is ALPHA (Amplified Luminescence Proximity Homogeneous Assay), is superior in versatility and quantitativity to other homogeneous assays, because it is not influenced by bonding sites between donor beads and acceptor beads, and uses excitation light with long wavelength, which is little exerted by interference caused by assay components (Non-patent document 1).
On the other hand, in order to efficiently obtain various proteins necessary for the above-mentioned assays, cell-free protein synthetic means are utilized these days. The methods have used rabbit reticulocyte cell-free systems (Reticulocyte Lysate) and Escherichia coli extract cell-free systems. However, wheat germ extract preparation methods, and high efficiency cell-free protein synthesis systems using the wheat germ extract, which are based on the clarification of destabilizing mechanism of wheat germ cell-free system (Wheat Germ Extract), and are stable and have high translation activity, are provided (non-patent document 2 and patent documents 1 to 3). Furthermore, screening methods using the wheat germ cell-free protein synthesizing system are provided (patent document 4).    [Non-patent document 1] a home page of PerkinElmer Japan Co., Ltd.    [Non-patent document 2] Proc. Natl. Acad. Sci. USA, 99: 14652-14657 (2002)    [Patent document 1] WO00/68412 A1    [Patent document 2] WO02/24939 A1    [Patent document 3] WO2005/063979 A1    [Patent document 4] WO2005/035780 A1
In various detection methods, biotinylation is performed for labeling proteins (include stabilizing protein). Particularly, in the above-mentioned typical ALPHA product, ALPHASCREEN™ commercially available from PerkinElmer Co., Ltd., in order to analyze interaction between a protein and a biological molecule, a step for biotinylating the protein is essential. In various detection methods, however, presence of a large amount of free biotin derivatives in a reaction solution containing biotin-tagged protein exerts great influences on detection. A step of removing free biotin derivates is necessary, accordingly. Because of this, in various detection methods, particularly in detection steps of ALPHA, the free biotin-removing step before detection makes the operations of whole assay troublesome, and, as a result, they had problems in rapidly analyzing polyspecimen.